Inbred corn line I9545

ABSTRACT

An inbred corn line designated I9545 is disclosed. The invention relates to the plants and seeds of inbred corn line I9545 and methods for producing a corn plant produced by crossing the inbred corn line I9545 with itself or with another corn plant. The invention also relates to methods for producing a corn plant containing in its genetic material one or more transgenes and to the transgenic corn plants and plant parts produced by those methods. The invention also relates to corn cultivars and plant parts derived from inbred corn line I9545 and to methods for producing other corn cultivars, lines, or plant parts derived from inbred corn line I9545, and to the corn plants and parts derived from use of those methods. The invention further relates to hybrid corn seeds, plants, and plant parts produced by crossing inbred corn line I9545 with another corn cultivar.

BACKGROUND OF THE INVENTION

This present invention relates to a new and distinctive inbred dent cornline designated I9545. All publications cited in this application areherein incorporated by reference.

The goal of plant breeding is to combine in a single variety or hybridvarious desirable traits. For vegetable crops, such as sweet corn, thesetraits may include resistance to diseases and insects, tolerance to heatand drought, reducing the time to crop maturity, greater yield, betteragronomic quality, processing traits, such as high processing plantrecovery, tender kernels, pleasing taste, uniform kernel size and color,attractive husk package and husked ears, ability to ship long distances,ease of mechanical or manual harvest, tipfill, row straight. Withmechanical harvesting of many crops, uniformity of plant characteristicssuch as germination and stand establishment, growth rate, maturity, andplant and ear height, is important.

Corn (Zea mays L., also called maize) is the most valuable crop grown inthe United States. Along with wheat, rice, and potatoes, corn ranks asone of the four most important crops in the world. Three major types ofcorn are grown in the United States: 1) grain or field corn; 2) sweetcorn; and 3) popcorn. Grain or field corn is grown annually for grain onfrom 55 to 60 million acres, with seed production in excess of 4 billionbushels, and in addition, around 8 million acres of this type areharvested for silage. Grain corn is further classified commercially intofour main types: 1) dent corn; 2) flint corn; 3) flour or soft corn; and4) waxy corn.

Dent corn is a particular type of grain corn. Dent corn is the mostcommon type of corn, comprising about 90 percent of the corn grown inthe United States. Dent corn, when fully ripe, has a pronounceddepression or dent at the crown of the kernels. The kernels contain ahard form of starch at the sides and a soft type of starch in thecenter. This latter starch shrinks as the kernel ripens resulting in theterminal depression. Dent varieties vary in kernel shape from long andnarrow to wide and shallow. Farmers harvest dent corn when the seedsbecome hard and ripe. Dent corn is primarily used as a livestock feed,but can also be used to make many food and industrial products. Dentcorn is grown in all parts of the United States Corn Belt.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

SUMMARY OF THE INVENTION

In one aspect of the present invention, there is provided a novel inbreddent corn line, designated I9545. Thus, one aspect of this inventionrelates to the seeds of inbred corn line I9545, to the plants of inbredcorn line I9545 and parts thereof, for example pollen, ovule, or ear,and to methods for producing a maize plant, preferably a dent cornplant, by crossing the inbred line I9545 with itself or another maizeline. A further aspect relates to hybrid maize seeds, preferably hybridcorn seeds, and plants produced by crossing the inbred line I9545 withanother maize line.

Another aspect of the present invention is also directed to inbred cornline I9545 into which one or more specific, single gene traits, forexample transgenes, have been introgressed from another maize line, suchas a field corn line or a sweet corn line, and which has essentially allof the morphological and physiological characteristics of inbred cornline of I9545, in addition to the one or more specific, single genetraits introgressed into the inbred. Another aspect of the presentinvention also relates to seeds of an inbred corn line I9545 into whichone or more specific, single gene traits have been introgressed and toplants of an inbred corn line I9545 into which one or more specific,single gene traits have been introgressed. A further aspect of thepresent invention relates to methods for producing a maize plant,preferably a dent corn plant, by crossing plants of an inbred corn lineI9545 into which one or more specific, single gene traits have beenintrogressed with themselves or with another maize line.

Another aspect of the present invention relates to hybrid maize seeds,preferably dent corn seeds, and plants produced by crossing plants of aninbred corn line I9545 into which one or more specific, single genetraits have been introgressed with another maize line. A further aspectof the present invention is also directed to a method of producinginbreds comprising planting a collection of hybrid seed, growing plantsfrom the collection, identifying inbreds among the hybrid plants,selecting the inbred plants and controlling their pollination topreserve their homozygosity.

Another aspect of the present invention is also directed to a method ofproducing a corn product comprising obtaining an ear of a plantaccording to the instant invention, isolating a kernel from said ear,and processing said kernel to obtain a dent corn product. In a furtheraspect of the present invention, a corn product according the instantinvention is a livestock feed.

DETAILED DESCRIPTION OF THE INVENTION

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. The allele is any of one or more alternative form of a gene, allof which relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Collection of seeds. In the context of the present invention acollection of seeds will be a grouping of seeds mainly containingsimilar kinds of seeds, for example hybrid seeds having the inbred lineof the invention as a parental line, but that may also contain, mixedtogether with this first kind of seeds, a second, different kind ofseeds, of one of the inbred parent lines, for example the inbred line ofthe present invention. A commercial bag of hybrid seeds having theinbred line of the invention as a parental line and containing also theinbred line seeds of the invention would be, for example such acollection of seeds.

Crossing. The pollination of a female flower of a corn plant, therebyresulting in the production of seed from the flower.

Cross-pollination. Fertilization by the union of two gametes fromdifferent plants.

Daily heat unit value. The daily heat unit value is calculated asfollows: (the maximum daily temperature+the minimum daily temperature)/2minus 50. All temperatures are in degrees Fahrenheit. The maximumtemperature threshold is 86 degrees, if temperatures exceed this, 86 isused. The minimum temperature threshold is 50 degrees, if temperaturesgo below this, 50 is used.

Dent corn. Botanically known as Zea mays var. indentata. A tall-growingvariety of corn having yellow or white kernels that are indented at thetip.

Dropped ears. Ears that have fallen from the plant to the ground.

Dry down. This is the rate at which a hybrid will reach acceptableharvest moisture

Ear cob diameter. The average diameter of the cob measured at themidpoint.

Ear diameter. The average diameter of the ear at its midpoint.

Ear height. The ear height is a measure from the ground to the upper earnode attachment, and is measured in centimeters.

Ear length. The average length of the ear.

Ear shank length. The average length of the ear shank.

Ear taper (shape). The taper or shape of the ear scored as 1=slight,2=average, and 3=extreme.

Ear weight. The average weight of an ear.

Emasculate. The removal of plant male sex organs or the inactivation ofthe organs with a chemical agent or a cytoplasmic or nuclear geneticfactor conferring male sterility.

Endosperm type. Endosperm type refers to endosperm genes and types suchas starch, sugary alleles (su1, su2, etc.), sugary enhancer or extender,waxy, amylose extender, dull, brittle alleles (bt1, bt2, etc.) other sh2alleles, and any combination of these.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics, except for the characteristics derived from theconverted gene.

GDUs. Growing degree units which are calculated by the Barger Method,where the heat units for a 24 hour period are calculated asGDUs=[(Maximum daily temperature+Minimum daily temperature)/2]−50. Thehighest maximum daily temperature used is 86° F. and the lowest minimumtemperature used is 50° F.

GDUs to shed. The number of growing degree units (GDUs) or heat unitsrequired for a variety to have approximately 50% of the plants sheddingpollen as measured from time of planting. GDUs to shed is determined bysumming the individual GDU daily values from planting date to the dateof 50% pollen shed.

GDUs to silk. The number of growing degree units for a variety to haveapproximately 50% of the plants with silk emergence as measured fromtime of planting. GDUs to silk is determined by summing the individualGDU daily values from planting date to the date of 50% silking.

Herbicide resistant or tolerant. A plant containing anyherbicide-resistant gene or any DNA molecule or construct (or replicatethereof) which is not naturally occurring in the plant which results inincrease tolerance to any herbicide including, imidazoline,sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine andbenzonitrile. For purposes of this definition, a DNA molecule orconstruct shall be considered to be naturally occurring if it exists ina plant at a high enough frequency to provide herbicide resistancewithout further selection and/or if it has not been produced as a resultof tissue culture selection, mutagenesis, genetic engineering usingrecombinant DNA techniques or other in vitro or in vivo modification tothe plant.

HTU. HTU is the summation of the daily heat unit value calculated fromplanting to harvest.

Inbreeding depression. The inbreeding depression is the loss ofperformance of the inbreds due to the effect of inbreeding, i.e. due tothe mating of relatives or to self-pollination. It increases thehomozygous recessive alleles leading to plants which are weaker andsmaller and having other less desirable traits.

Kernel aleurone color. The color of the aleurone scored as white, pink,tan, brown, bronze, red, purple, pale purple, colorless, or variegated.

Kernel length. The average distance from the cap of the kernel to thepedicel.

Moisture. The moisture is the actual percentage moisture of the grain atharvest.

Plant cell. Plant cell, as used herein includes plant cells whetherisolated, in tissue culture, or incorporated in a plant or plant part.

Plant habit. This is a visual assessment assigned during the latevegetative to early reproductive stages to characterize the plants leafhabit. It ranges from decumbent with leaves growing horizontally fromthe stalk to a very upright leaf habit, with leaves growing nearvertically from the stalk.

Plant height. This is a measure of the height of the hybrid from theground to the tip of the tassel, and is measured in centimeters.

Plant intactness. This is a visual assessment assigned to a hybrid orinbred at or close to harvest to indicate the degree that the plant hassuffered disintegration through the growing season. Plants are ratedfrom 1 (poorest) to 9 (best) with poorer scores given for plants thathave more of their leaf blades missing.

Plant part. As used herein, the term “plant part” includes leaves,stems, roots, seeds, grains, embryos, pollens, ovules, flowers, ears,cobs, husks, stalks, root tips, anthers, silk, tissue, cells and thelike.

Pollen shed. This is a visual rating assigned at flowering to describethe abundance of pollen produced by the anthers. Inbreds are rated 1(poorest) to 9 (best) with the best scores for inbreds with tassels thatshed more pollen during anthesis.

Post-anthesis root lodging. This is a percentage plants that root lodgeafter anthesis: plants that lean from the vertical axis at anapproximately 30° angle or greater.

Pre-anthesis brittle snapping. This is a percentage of “snapped” plantsfollowing severe winds prior to anthesis

Pre-anthesis root lodging. This is a percentage plants that root lodgeprior to anthesis: plants that lean from the vertical axis at anapproximately 30° angle or greater.

Quantitative Trait Loci (QTL) Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Root Lodging. The root lodging is the percentage of plants that rootlodge (i.e., those that lean from the vertical axis at an approximate300 angle or greater would be counted as root lodged).

Seed quality. This is a visual rating assigned to the kernels of theinbred. Kernels are rated 1 (poorest) to 9 (best) with poorer scoresgiven for kernels that are very soft and shriveled with splitting of thepericarp visible and better scores for fully formed kernels.

Seedling vigor. This is the vegetative growth after emergence at theseedling stage, approximately five leaves.

Silking ability. This is a visual assessment given during flowering.Plants are rated on the amount and timing of silk production. Plants arerated from 1 (poorest) to 9 (best) with poorer scores given for plantsthat produce very little silks that are delayed past pollen shed.

Single gene converted. Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of an inbred are recovered in addition tothe single gene transferred into the inbred via the backcrossingtechnique or via genetic engineering.

Stalk lodging. This is the percentage of plants that stalk lodge, i.e.,stalk breakage, as measured by either natural lodging or pushing thestalks and determining the percentage of plants that break off below theear. This is a relative rating of an inbred to other inbreds, or ahybrid to other hybrids for standability.

Standability. A term referring to the how well a plant remains uprighttowards the end of the growing season. Plants with excessive stalkbreakage and/or root lodging would be considered to have poorstandability.

Stay Green. Stay green is the measure of plant health near the time ofblack layer formation (physiological maturity). A high score indicatesbetter late-season plant health.

Transgene. A genetic sequence which has been introduced into the nuclearor chloroplast genome of a corn plant by a genetic transformationtechnique.

Variety. A plant variety as used by one skilled in the art of plantbreeding means a plant grouping within a single botanical taxon of thelowest known rank which can be defined by the expression of thecharacteristics resulting from a given genotype or combination ofphenotypes, distinguished from any other plant grouping by theexpression of at least one of the said characteristics and considered asa unit with regard to its suitability for being propagated unchanged(International convention for the protection of new varieties of plants)

Yield. The yield is the tons of green corn or green weight per acre. Itcan also be defined as the number of ears per acre or per plant.

Yield (Bushels/Acre). The yield is the actual yield of the grain atharvest adjusted to 15.5% moisture.

According to the invention, there is provided a novel inbred corn line,designated I9545. Inbred corn line I9545 was developed in a breedingprogram aimed at developing a breeding line with good agronomiccharacteristics, such as standability, disease resistance, seed yieldand utility as a male in seed production. I9545 was selected based on a“per se” basis by evaluating its performance in hybrid combinations andselecting for grain yield, grain test weight, root and stalk strength.

The inbred has shown uniformity and stability as described in thefollowing variety description information. It has been self-pollinated asufficient number of generations with careful attention to plant type.The line has been increased with continued observation for uniformity.Inbred corn line I9545 has the following morphological and othercharacteristics based on data taken in 2012 in Sharpesville and Atlanta,Ind.

TABLE 1 VARIETY DESCRIPTION INFORMATION TYPE: Dent corn REGION WHEREDEVELOPED IN THE U.S.A.: Eastern corn belt MATURITY: From emergence to50% of plants in silk: Days: 62 Heat units: 1573 From emergence to 50%of plant in pollen: Days: 60 Heat units: 1501 PLANT: Plant height (totassel tip) (inches): 72.0 Ear height (to base of top ear node)(inches): 28.0 Length of top ear internode (inches): 10.0 Average numberof tillers: 0.1 Average number of ears per stalk: 2 Anthocyanin of braceroots: Faint LEAF: Width of ear node leaf (cm): 8.89 Length of ear nodeleaf (cm): 64.1 Number of leaves above top ear: 4 Degree leaf angle(from 2^(nd) leaf above ear at anthesis to stalk above leaf): 35 Leafcolor: Moderate green Leaf sheath pubescence (1 = none, 9 = like peachfuzz): 3 Marginal waves (1 = none, 9 = many): 8 Longitudinal creases (1= none, 9 = many): 3 TASSEL: Color: Green Number of primary lateralbranches: 5 to 6 Branch angle from central spike: 30 Tassel length (fromtop leaf collar to tassel tip) (cm): 48.3 Pollen shed (0 = male sterile,9 = heavy shed): 6 Anther color: Green Glume color: Green EAR: Unhuskeddata: Silk color (3 days after emergence): Pink Fresh husk color (25days after 50% silking): Light green Dry husk color (65 days after 50%silking): Light brown/Tan Position of ear at dry husk stage (1 =upright, 2 = horizontal, 3 = pendent): 1 Husk tightness (1 = very loose,9 = very tight): 6 Husk extension (at harvest) (1 = short (earsexposed), 2 = medium (<8 cm), 3 = long (8-10 cm beyond ear tip), 4 =very long (>10 cm)): 2 Husked ear data: Ear length (cm): 16.1 Eardiameter at mid-point (inches): 2.54 Ear weight (gm): 136.93 gm at 10.3%moisture Number of kernel rows: 16 Kernel rows (1 = indistinct, 2 =distinct): 2 Row alignment (1 = straight, 2 = slightly curved, 3 =spiral): 2 Shank length (cm): 13.2 Ear taper (1 = slight, 2 = average, 3= extreme): 1 KERNEL (Dried): Kernel length (mm): 9.5 Kernel width (mm):6.35 Kernel thickness (mm): 3.2 % Round kernels (shape grade): 67%Aleurone color pattern (1 = homozygous, 2 = segregating): 1 Aleuronecolor: Absent Hard endosperm color: White Endosperm type (1 = sweet(su1), 2 = extra sweet (sh2), 3 = normal starch, 4 = high 3 amylosestarch, 5 = waxy starch, 6 = highpProtein, 7 = high lysine, 8 = supersweet (se), 9 = high oil, 10 = other): Weight per 100 kernels (unsizedsample) (gm): 29.7 COB: Cob diameter at mid-point (inches): 1.0 Cobcolor: Red DISEASE RESISTANCE (1 = most susceptible, 9 = mostresistant): Leaf blights, wilts, and local infection diseases: Commonrust (Puccinia sorghi): 7 Common smut (Ustilago maydis): 8 Eyespot(Kabatiella zeae): 8 Goss's wilt (Clavibacter michiganense spp.nebraskense): 8 Gray leaf spot (Cercospora zeae-maydis): 8Helminthosporium leaf spot (Bipolaris zeicola): 7 Northern leaf blight(Exserohilum turcicum): 7 Stewart's wilt (Erwinia stewartii): 7 Systemicdiseases: Corn lethal necrosis (MCMV and MDMV): 8 Head smut(Sphacelotheca reiliana): 7 Stalk rots: Anthracnose stalk rot(Colletotrichum graminicola): 5 Diplodia stalk rot (Stenocarpellamaydis): 6 Fusarium stalk rot (Fusarium moniliforme): 6 Gibberella stalkrot (Gibberella zeae): 6 Ear and kernel rots: Aspergillus ear and kernelrot (Aspergillus flavus): 4 Diplodia ear rot (Stenocarpella maydis): 4Fusarium ear and kernel rot (Fusarium moniliforme): 4 Gibberella ear rot(Gibberella zeae): 5 PEST RESISTANCE (1 = most susceptible, 9 = mostresistant): Corn earworm (Helicoverpa zea): 1 Leaf-feeding: 3Silk-feeding: 3 Ear damage: 7 European corn borer (Ostrinia nubilalis):4 1^(st) generation (typically whorl leaf feeding): 4 2^(nd) generation(typically leaf sheath-collar feeding): 4 Fall armyworm (Spodopterafrugiperda): Leaf-feeding: 4 Silk-feeding: 4

Further Embodiments of Invention

Dent corn is an important and valuable vegetable crop. Thus, acontinuing goal of plant breeders is to develop high-yielding hybridsthat are agronomically sound based on stable inbred lines. The reasonsfor this goal are obvious: to maximize the amount of marketable dentcorn produced with the inputs used and minimize susceptibility of thecrop to pests and environmental stresses.

To accomplish this goal, the breeder must select and develop superiorinbred parental lines for producing hybrids. This requiresidentification and selection of genetically unique individuals thatoccur in a segregating population. The segregating population is theresult of a combination of crossover events plus the independentassortment of specific combinations of alleles at many gene loci thatresults in specific genotypes. The probability of selecting any oneindividual with a specific genotype from a breeding cross is very lowdue to the large number of segregating genes and the unlimitedrecombinations of these genes, some of which may be closely linked.However, the genetic variation among individual progeny of a breedingcross allows for the identification of rare and valuable new genotypes.These new genotypes are neither predictable nor incremental in value,but rather the result of manifested genetic variation combined withselection methods, environments and the actions of the breeder.

Thus, even if the entire genotypes of the parents of the breeding crosswere characterized and a desired genotype known, only a few, if any,individuals having the desired genotype may be found in a largesegregating F₂ population. Typically, however, neither the genotypes ofthe breeding cross parents nor the desired genotype to be selected isknown in any detail. In addition, it is not known how the desiredgenotype would react with the environment. This genotype by environmentinteraction is an important, yet unpredictable, factor in plantbreeding. A breeder of ordinary skill in the art cannot predict thegenotype, how that genotype will interact with various climaticconditions or the resulting phenotypes of the developing lines, exceptperhaps in a very broad and general fashion. A breeder of ordinary skillin the art would also be unable to recreate the same line twice from thevery same original parents as the breeder is unable to direct how thegenomes combine or how they will interact with the environmentalconditions. This unpredictability results in the expenditure of largeamounts of research resources in the development of a superior new maizeinbred line, such as a superior new sweet corn inbred line.

Inbred maize lines, such as inbred corn lines, are typically developedfor use in the production of hybrid maize lines, for example hybrid dentcorn lines. Inbred maize lines need to be highly homogeneous, homozygousand reproducible to be useful as parents of commercial hybrids. Thereare many analytical methods available to determine the homozygotic andphenotypic stability of these inbred lines. The oldest and mosttraditional method of analysis is the observation of phenotypic traits.The data is usually collected in field experiments over the life of themaize plants to be examined. Phenotypic characteristics often observedare for traits associated with plant morphology, ear and kernelmorphology, insect and disease resistance, maturity, and yield.

In addition to phenotypic observations, the genotype of a plant can alsobe examined. There are many laboratory-based techniques available forthe analysis, comparison and characterization of plant genotype; amongthese are Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats(SSRs) which are also referred to as Microsatellites, and SingleNucleotide Polymorphisms (SNPs).

Some of the most widely used of these laboratory techniques are IsozymeElectrophoresis and RFLPs as discussed in Lee, M., “Inbred Lines ofMaize and Their Molecular Markers,” The Maize Handbook, Springer-Verlag,New York, Inc., pp. 423-432 (1994) incorporated herein by reference.Isozyme Electrophoresis is a useful tool in determining geneticcomposition, although it has relatively low number of available markersand the low number of allelic variants among maize inbreds. RFLPs havethe advantage of revealing an exceptionally high degree of allelicvariation in maize and the number of available markers is almostlimitless. Maize RFLP linkage maps have been rapidly constructed andwidely implemented in genetic studies. One such study is described inBoppenmaier, et al., “Comparisons among strains of inbreds for RFLPs,”Maize Genetics Cooperative Newsletter, 65:1991, p. 90, is incorporatedherein by reference. This study used 101 RFLP markers to analyze thepatterns of two to three different deposits each of five differentinbred lines. The inbred lines had been selfed from nine to 12 timesbefore being adopted into two to three different breeding programs. Itwas results from these two to three different breeding programs thatsupplied the different deposits for analysis. These five lines weremaintained in the separate breeding programs by selfing or sibbing androgueing off-type plants for an additional one to eight generations.After the RFLP analysis was completed, it was determined the five linesshowed 0-2% residual heterozygosity. Although this was a relativelysmall study, it can be seen using RFLPs that the lines had been highlyhomozygous prior to the separate strain maintenance.

The laboratory-based techniques described above, in particular RFLP andSSR, are routinely used in such backcrosses to identify the progenieshaving the highest degree of genetic identity with the recurrent parent.This permits to accelerate the production of inbred maize lines havingat least 90%, preferably at least 95%, more preferably at least 99%genetic identity with the recurrent parent, yet more preferablygenetically identical to the recurrent parent, except for the trait(s)introgressed from the donor patent. Such determination of geneticidentity is based on molecular markers used in the laboratory-basedtechniques described above. Such molecular markers are for example thosedescribed in Boppenmaier, et al., “Comparisons among strains of inbredsfor RFLPs,” Maize Genetics Cooperative Newsletter 65, p. 90 (1991),incorporated herein by reference, or those available from the Universityof Missouri database and the Brookhaven laboratory database (seehttp://www.agron.missouri.edu, incorporated herein by reference). Thelast backcross generation is then selfed to give pure breeding progenyfor the gene(s) being transferred. The resulting plants have essentiallyall of the morphological and physiological characteristics of inbredcorn line I9545, in addition to the single gene trait(s) transferred tothe inbred. The exact backcrossing protocol will depend on the traitbeing altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the trait being transferred isa dominant allele, a recessive allele may also be transferred. In thisinstance it may be necessary to introduce a test of the progeny todetermine if the desired trait has been successfully transferred.

Maize is bred through techniques that take advantage of the plant'smethod of pollination. A plant is self-pollinated if pollen from oneflower is transferred to the same or another flower of the same plant. Aplant is cross-pollinated if the pollen comes from a flower on adifferent plant. Plants that have been self-pollinated and selected fortype for many generations become homozygous at almost all gene loci andproduce a uniform population of true breeding progeny. A cross betweentwo different homozygous lines produces a uniform population of hybridplants that may be heterozygous for many gene loci. A cross of twoplants each heterozygous at a number of gene loci will produce apopulation of hybrid plants that differ genetically and will not beuniform.

Maize can be bred by both self-pollination and cross-pollinationtechniques. Maize has separate male and female flowers on the sameplant, located on the tassel and the ear, respectively. Naturalpollination occurs in maize when wind blows pollen from the tassels tothe silks that protrude from the tops of the ears.

Pedigree breeding starts with the crossing of two genotypes, each ofwhich may have one or more desirable characteristics that is lacking inthe other or which complements the other. If the two original parents donot provide all the desired characteristics, other sources can beincluded in the breeding population. In the pedigree method, superiorplants are selfed and selected in successive generations. In thesucceeding generations the heterozygous condition gives way tohomogeneous lines as a result of self-pollination and selection.Typically in the pedigree method of breeding five or more generations ofselfing and selection is practiced: F₁ to F₂; F₃ to F₄; F₄ to F₅, etc.

Recurrent selection breeding can be used to improve populations ofeither self or cross-pollinating crops. Recurrent selection can be usedto transfer a specific desirable trait from one inbred or source to aninbred that lacks the trait. This can be accomplished, for example, byfirst a superior inbred (recurrent parent) to a donor inbred(non-recurrent parent), that carries the appropriate gene(s) for thetrait in question. The progeny of this cross is then mated back to thesuperior recurrent parent followed by selection in the resultant progenyfor the desired trait to be transferred from the non-recurrent parent.After five or more backcross generations with selection for the desiredtrait, the progeny will be homozygous for loci controlling thecharacteristic being transferred, but will be like the superior parentfor essentially all other genes. The last backcross generation is thenselfed to give pure breeding progeny for the gene(s) being transferred.A hybrid developed from inbreds containing the transferred gene(s) isessentially the same as a hybrid developed from the same inbreds withoutthe transferred genes, except for the difference made by the transferredgene. As the varieties developed using recurrent selection breedingcontain almost all of the characteristics of the recurrent parent,selecting a superior recurrent parent is desirable.

Maize Hybrid Development

In another embodiment of the invention, one or both first and secondparent corn plants can be from variety I9545. Thus, any corn plantproduced using corn plant I9545 forms a part of the invention. As usedherein, crossing can mean selfing, backcrossing, crossing to another orthe same variety, crossing to populations, and the like. All corn plantsproduced using the corn variety I9545 as a parent are, therefore, withinthe scope of this invention.

One use of the instant corn variety is in the production of hybrid seed.Any time the corn plant I9545 is crossed with another, different, cornplant, a corn hybrid plant is produced. As such, hybrid corn plant canbe produced by crossing I9545 with any second corn plant. Essentiallyany other corn plant can be used to produce a corn plant having cornplant I9545 as one parent. All that is required is that the second plantbe fertile, which corn plants naturally are, and that the plant is notcorn variety I9545.

The goal of the process of producing an F₁ hybrid is to manipulate thegenetic complement of corn to generate new combinations of genes whichinteract to yield new or improved traits (phenotypic characteristics). Aprocess of producing an F₁ hybrid typically begins with the productionof one or more inbred plants. Those plants are produced by repeatedcrossing of ancestrally related corn plants to try to combine certaingenes within the inbred plants.

When the corn plant I9545 is crossed with another plant to yieldprogeny, it can serve as either the maternal or paternal plant. For manycrosses, the outcome is the same regardless of the assigned sex of theparental plants. However, due to increased seed yield and productioncharacteristics, it may be desired to use one parental plant as thematernal plant. Some plants produce tighter ear husks leading to moreloss, for example due to rot. There can be delays in silk formationwhich deleteriously affect timing of the reproductive cycle for a pairof parental inbreds. Seed coat characteristics can be preferable in oneplant. Pollen can be shed better by one plant. Other variables can alsoaffect preferred sexual assignment of a particular cross.

The development of a maize hybrid involves three steps: (1) theselection of plants from various germplasm pools for initial breedingcrosses; (2) the selfing of the selected plants from the breedingcrosses for several generations to produce a series of inbred lines,which, although different from each other, breed true and are highlyuniform; and (3) crossing the selected inbred lines with differentinbred lines to produce the hybrid progeny (F₁). During the inbreedingprocess in maize, the vigor of the lines decreases. Vigor is restoredwhen two different inbred lines are crossed to produce the hybridprogeny (F₁). An important consequence of the homozygosity andhomogeneity of the inbred lines is that the hybrid between a definedpair of inbreds will always have the same genotype. Once the inbredsthat give a superior hybrid have been identified, the hybrid seed can bereproduced indefinitely as long as the homogeneity of the inbred parentsis maintained.

A single cross maize hybrid results from the cross of two inbred lines,each of which has a genotype that complements the genotype of the other.The hybrid progeny of the first generation is designated F₁. In thedevelopment of commercial hybrids only the F₁ hybrid plants are sought.Preferred F₁ hybrids are more vigorous than their inbred parents. Thishybrid vigor, or heterosis, can be manifested in many polygenic traits,including increased vegetative growth and increased yield.

A single cross hybrid is produced when two inbred lines are crossed toproduce the F₁ progeny. A double cross hybrid is produced from fourinbred lines crossed in pairs (A×B and C×D) and then the two F₁ hybridsare crossed again (A×B)×(C×D). Much of the hybrid vigor exhibited by F₁hybrids is lost in the next generation (F₂). Consequently, seed fromhybrids is not used for planting stock.

A reliable method of controlling male fertility in plants offers theopportunity for improved plant breeding. This is especially true fordevelopment of maize hybrids, which relies upon some sort of malesterility system. There are several options for controlling malefertility available to breeders, such as: manual or mechanicalemasculation (or detasseling), cytoplasmic male sterility, genetic malesterility, gametocides and the like.

Hybrid maize seed is typically produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twomaize inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Providing that there issufficient isolation from sources of foreign maize pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

Another system useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see, Carlson, Glenn R., U.S. Pat. No. 4,936,904). Applicationof the gametocide, timing of the application and genotype specificityoften limit the usefulness of the approach.

The use of male sterile inbreds is but one factor in the production ofmaize hybrids. The development of maize hybrids requires, in general,the development of homozygous inbred lines, the crossing of these lines,and the evaluation of the crosses. Pedigree breeding and recurrentselection breeding methods are used to develop inbred lines frombreeding populations. Breeding programs combine the genetic backgroundsfrom two or more inbred lines or various other germplasm sources intobreeding pools from which new inbred lines are developed by selfing andselection of desired phenotypes. The new inbreds are crossed with otherinbred lines and the hybrids from these crosses are evaluated todetermine which of those have commercial potential. Plant breeding andhybrid development are expensive and time consuming processes.

Hybrid seed production requires elimination or inactivation of pollenproduced by the female parent. Incomplete removal or inactivation of thepollen provides the potential for self pollination. This inadvertentlyself pollinated seed may be unintentionally harvested and packaged withhybrid seed. Once the seed is planted, it is possible to identify andselect these self pollinated plants. These self pollinated plants willbe genetically equivalent to the female inbred line used to produce thehybrid. Typically these self pollinated plants can be identified andselected due to their decreased vigor. Female selfs are identified bytheir less vigorous appearance for vegetative and/or reproductivecharacteristics, including shorter plant height, small ear size, ear andkernel shape, cob color, or other characteristics.

Identification of these self pollinated lines can also be accomplishedthrough molecular marker analyses. See, “The Identification of FemaleSelfs in Hybrid Maize: A Comparison Using Electrophoresis andMorphology,” Smith, J. S. C. and Wych, R. D., Seed Science andTechnology 14, pp. 1-8 (1995), the disclosure of which is expresslyincorporated herein by reference. Through these technologies, thehomozygosity of the self pollinated line can be verified by analyzingallelic composition at various loci along the genome. Those methodsallow for rapid identification of the invention disclosed herein. Seealso, “Identification of Atypical Plants in Hybrid Maize Seed byPostcontrol and Electrophoresis,” Sarca, V., et al., Probleme deGenetica Teoretica si Aplicata, Vol. 20 (1), pp. 29-42.

As is readily apparent to one skilled in the art, the foregoing are onlytwo of the various ways by which the inbred can be obtained by thoselooking to use the germplasm. Other means are available, and the aboveexamples are illustrative only.

Introduction of a New Trait or Locus into Inbred Corn I9545

The invention also encompasses plants of inbred corn line I9545 andparts thereof further comprising one or more specific, single genetraits, which have been introgressed into inbred corn line I9545 fromanother maize line. The single gene traits is transferred into inbredcorn line I9545 from any type of maize line, such as for example a fieldcorn line, a sweet corn line, a popcorn line, a white corn line or asilage corn line. Preferably, one or more new traits are transferred toinbred corn line I9545, or, alternatively, one or more traits of inbredcorn line I9545 are altered or substituted. The transfer (orintrogression) of the trait(s) into inbred corn line I9545 is forexample achieved by recurrent selection breeding, for example bybackcrossing. In this case, inbred corn line I9545 (the recurrentparent) is first crossed to a donor inbred (the non-recurrent parent)that carries the appropriate gene(s) for the trait(s) in question. Theprogeny of this cross is then mated back to the recurrent parentfollowed by selection in the resultant progeny for the desired trait(s)to be transferred from the non-recurrent parent. After three, preferablyfour, more preferably five or more generations of backcrosses with therecurrent parent with selection for the desired trait(s), the progenywill be heterozygous for loci controlling the trait(s) beingtransferred, but will be like the recurrent parent for most or almostall other genes (see, for example, Poehlman & Sleper, Breeding FieldCrops, 4th Ed., 172-175 (1995); Fehr, Principles of CultivarDevelopment, Vol. 1: Theory and Technique, 360-376 (1987), incorporatedherein by reference).

Many traits have been identified that are not regularly selected for inthe development of a new inbred but that can be improved by backcrossingtechniques. Examples of traits transferred to inbred corn line I9545include, but are not limited to, waxy starch, herbicide tolerance,resistance for bacterial, fungal, or viral disease, insect resistance,enhanced nutritional quality, improved performance in an industrialprocess, quality and processing traits such as high processing plantrecovery, tender kernels, pleasing taste, uniform kernel size and color,attractive husk package and husked ears, ability to ship long distances,ease of mechanical or manual harvest, tipfill, row straight, alteredreproductive capability, such as male sterility or male fertility, yieldstability and yield enhancement. Other traits transferred to inbred cornline I9545 are for the production of commercially valuable enzymes ormetabolites in plants of inbred corn line I9545.

Traits transferred to inbred corn line I9545 are naturally occurringmaize traits, such as naturally occurring dent corn traits, or aretransgenic. Transgenes are originally introduced into a donor,non-recurrent parent using genetic engineering and transformationtechniques well known in the art. A transgene introgressed into inbredcorn line I9545 typically comprises a nucleotide sequence whoseexpression is responsible or contributes to the trait under the controlof a promoter appropriate for the expression of the nucleotide sequenceat the desired time in the desired tissue or part of the plant.Constitutive or inducible promoters are used. The transgene may alsocomprise other regulatory elements such as for example translationenhancers or termination signals. In one embodiment, the nucleotidesequence is the coding sequence of a gene and is transcribed andtranslated into a protein. In another embodiment, the nucleotidesequence encodes an antisense RNA or a sense RNA that is not translatedor only partially translated.

Where more than one trait is introgressed into inbred corn line I9545,it is preferred that the specific genes are all located at the samegenomic locus in the donor, non-recurrent parent, preferably, in thecase of transgenes, as part of a single DNA construct integrated intothe donor's genome. Alternatively, if the genes are located at differentgenomic loci in the donor, non-recurrent parent, backcrossing allowsrecovery of all of the morphological and physiological characteristicsof inbred corn line I9545 in addition to the multiple genes in theresulting sweet corn inbred line.

The genes responsible for a specific, single gene trait are generallyinherited through the nucleus. Known exceptions are, e.g., the genes formale sterility, some of which are inherited cytoplasmically, but stillact as single gene traits. In one embodiment, a transgene to beintrogressed into inbred corn line I9545 is integrated into the nucleargenome of the donor, non-recurrent parent. In another embodiment, atransgene to be introgressed into inbred corn line I9545 is integratedinto the plastid genome of the donor, non-recurrent parent. In oneembodiment, a plastid transgene comprises one gene transcribed from asingle promoter or two or more genes transcribed from a single promoter.

In one embodiment, a transgene whose expression results or contributesto a desired trait to be transferred to inbred corn line I9545 comprisesa virus resistance trait such as, for example, a MDMV strain B coatprotein gene whose expression confers resistance to mixed infections ofmaize dwarf mosaic virus and maize chlorotic mottle virus in transgenicmaize plants (Murry, et al., Biotechnology 11:1559-64 (1993),incorporated herein by reference). In another embodiment, a transgenecomprises a gene encoding an insecticidal protein, such as, for example,a crystal protein of Bacillus thuringiensis or a vegetative insecticidalprotein from Bacillus cereus, such as VIP3 (see, for example, Estruch,et al., Nat Biotechnol 15:137-41 (1997), incorporated herein byreference). In one embodiment, an insecticidal gene introduced intoinbred corn line I9545 is a Cry1Ab gene or a portion thereof, forexample introgressed into inbred corn line I9545 from a maize linecomprising a Bt-11 event as described in U.S. application Ser. No.09/042,426, incorporated herein by reference, or from a maize linecomprising a 176 event as described in Koziel, et al., Biotechnology 11:194-200 (1993), incorporated herein by reference. In yet anotherembodiment, a transgene introgressed into inbred corn line I9545comprises an herbicide tolerance gene. For example, expression of analtered acetohydroxyacid synthase (AHAS) enzyme confers upon plantstolerance to various imidazolinone or sulfonamide herbicides (U.S. Pat.No. 4,761,373, incorporated herein by reference).

In another embodiment, a non-transgenic trait conferring tolerance toimidazolinones is introgressed into inbred corn line I9545 (e.g., an“IT” or “IR” trait). U.S. Pat. No. 4,975,374, incorporated herein byreference, relates to plant cells and plants containing a gene encodinga mutant glutamine synthetase (GS) resistant to inhibition by herbicidesthat are known to inhibit GS, e.g., phosphinothricin and methioninesulfoximine. Also, expression of a Streptomyces bar gene encoding aphosphinothricin acetyl transferase in maize plants results in toleranceto the herbicide phosphinothricin or glufosinate (U.S. Pat. No.5,489,520, incorporated herein by reference). U.S. Pat. No. 5,013,659,incorporated herein by reference, is directed to plants that express amutant acetolactate synthase (ALS) that renders the plants resistant toinhibition by sulfonylurea herbicides. U.S. Pat. No. 5,162,602,incorporated herein by reference, discloses plants tolerant toinhibition by cyclohexanedione and aryloxyphenoxypropanoic acidherbicides, such as, e.g., Sethoxydim or any herbicidally effectiveforms of2-[1-ethoxyimino)butyl]-5-(2-(ethylthio)propyl]-3-hydroxy-2-cyclohexen-1-one,its salts and derivatives. The tolerance is conferred by an alteredacetyl coenzyme A carboxylase (ACCase). U.S. Pat. No. 5,554,798,incorporated herein by reference, discloses transgenic glyphosatetolerant maize plants, which tolerance is conferred by an altered5-enolpyruvyl-3-phosphoshikimate (EPSP) synthase gene. Also, toleranceto a protoporphyrinogen oxidase inhibitor is achieved by expression of atolerant protoporphyrinogen oxidase enzyme in plants (U.S. Pat. No.5,767,373, incorporated herein by reference).

In one embodiment, a transgene introgressed into inbred corn line I9545comprises a gene conferring tolerance to an herbicide and at leastanother nucleotide sequence encoding another trait, such as for example,an insecticidal protein. Such combination of single gene traits is forexample a Cry1Ab gene and a bar gene.

Specific transgenic events introgressed into inbred corn line 19545 arefound at www.aphis.usda.gov/bbep/brs/not_reg.html incorporated herein byreference. These are for example introgressed from glyphosate tolerantevent GA21 (application number 9709901p), glyphosatetolerant/Lepidopteran insect resistant event MON 802 (application number9631701p), Lepidopteran insect resistant event DBT418(application number9629101p), male sterile event MS3 (application number 9522801p),Lepidopteran insect resistant event Bt11 (application number 9519501p),phosphinothricin tolerant event B16 (application number 9514501p),Lepidopteran insect resistant event MON 80100 (application number9509301p), phosphinothricin tolerant events T14, T25 (application number9435701p), Lepidopteran insect resistant event 176 (application number9431901p).

The introgression of a Bt11 event into a maize line, such as inbred cornline I9545, by backcrossing is exemplified in U.S. Pat. No. 6,114,608,incorporated herein by reference.

Direct selection may be applied where the trait acts as a dominanttrait. An example of a dominant trait is herbicide tolerance. For thisselection process, the progeny of the initial cross are sprayed with theherbicide prior to the backcrossing. The spraying eliminates any plantswhich do not have the desired herbicide tolerance characteristic, andonly those plants which have the herbicide tolerance gene are used inthe subsequent backcross. This process is then repeated for theadditional backcross generations.

This invention also is directed to methods for producing a maize plant,preferably a dent corn plant, by crossing a first parent maize plantwith a second parent maize plant wherein either the first or secondparent maize plant is a corn plant of inbred line I9545 or a corn plantof inbred line I9545 further comprising one or more single gene traits.Further, both first and second parent maize plants can come from theinbred corn line I9545 or an inbred corn plant of I9545 furthercomprising one or more single gene traits. Thus, any such methods usingthe inbred corn line I9545 or an inbred corn plant of I9545 furthercomprising one or more single gene traits are part of this invention:selfing, backcrosses, hybrid production, crosses to populations, and thelike. All plants produced using inbred corn line I9545 or inbred cornplants of I9545 further comprising one or more single gene traits as aparent are within the scope of this invention. Advantageously, inbredcorn line I9545 or inbred corn plants of I9545 further comprising one ormore single gene traits are used in crosses with other, different, maizeinbreds to produce first generation (F₁) maize hybrid seeds and plantswith superior characteristics.

In one embodiment, seeds of inbred corn line I9545 or seeds of inbredcorn plants of I9545 further comprising one or more single gene traitsare provided as an essentially homogeneous population of inbred cornseeds. Essentially homogeneous populations of inbred seed are those thatconsist essentially of the particular inbred seed, and are generallypurified free from substantial numbers of other seed, so that the inbredseed forms between about 90% and about 100% of the total seed, andpreferably, between about 95% and about 100% of the total seed. Mostpreferably, an essentially homogeneous population of inbred corn seedwill contain between about 98.5%, 99%, 99.5% and about 100% of inbredseed, as measured by seed grow outs. The population of inbred corn seedsof the invention is further particularly defined as being essentiallyfree from hybrid seed. Thus, one particular embodiment of this inventionis isolated inbred seed of inbred corn plants of I9545, e.g.,substantially free from hybrid seed or seed of other inbred seed, e.g.,a seed lot or unit of inbred seed which is at least 95% homogeneous. Theinbred seed population may be separately grown to provide an essentiallyhomogeneous population of plants of inbred corn line I9545 or inbredcorn plants of I9545 further comprising one or more single gene traits.

Seeds of inbred corn plants of I9545 for planting purposes is preferablycontainerized, e.g., placed in a bag or other container for ease ofhandling and transport and is preferably coated, e.g., with protectiveagents, e.g., safening or pesticidal agents, in particular antifungalagents and/or insecticidal agents.

When inbred corn line I9545 is identified herein, it is understood thatthe named line include varieties which have the same genotypic andphenotypic characteristics as the identified varieties, i.e., arederived from a common inbred source, even if differently named.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which maize plants can beregenerated, plant calli, plant clumps, and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,kernels, ears, cobs, leaves, husks, stalks, roots, root tips, anthers,silk, seeds and the like.

Origin and Breeding History of an Exemplary Introduced Trait

Provided by the invention are hybrid plants in which one or more of theparents comprise an introduced trait. Such a plant may be defined ascomprising a single locus conversion. Exemplary procedures for thepreparation of such single locus conversions are disclosed in U.S. Pat.No. 7,205,460, the entire disclosure of which is specificallyincorporated herein by reference.

As described, techniques for the production of corn plants with addedtraits are well known in the art (see, e.g., Poehlman, et al. (1995);Fehr (1987); Sprague and Dudley (1988)). A non-limiting example of sucha procedure one of skill in the art could use for preparation of ahybrid corn plant I9545 comprising an added trait is as follows:

-   -   (a) crossing a parent of hybrid corn plant I9545 to a second        (nonrecurrent) corn plant comprising a locus to be converted in        the parent;    -   (b) selecting at least a first progeny plant resulting from the        crossing and comprising the locus;    -   (c) crossing the selected progeny to the parent line of corn        plant I9545;    -   (d) repeating steps (b) and (c) until a parent line of variety        I9545 is obtained comprising the locus; and    -   (e) crossing the converted parent with the second parent to        produce hybrid variety I9545 comprising a desired trait.

Following these steps, essentially any locus may be introduced intohybrid corn variety I9545. For example, molecular techniques allowintroduction of any given locus, without the need for phenotypicscreening of progeny during the backcrossing steps. PCR and Southernhybridization are two examples of molecular techniques that may be usedfor confirmation of the presence of a given locus and thus conversion ofthat locus.

The seed of inbred corn line I9545 or of inbred corn line I9545 furthercomprising one or more single gene traits, the plant produced from theinbred seed, the hybrid maize plant produced from the crossing of theinbred, hybrid seed, and various parts of the hybrid maize plant can beutilized for human food, livestock feed, and as a raw material inindustry.

Corn Transformation

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed inbred line. An embodiment of the presentinvention comprises at least one transformation event.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). The expression vector maycontain one or more such operably linked gene/regulatory elementcombinations. The vector(s) may be in the form of a plasmid, and can beused alone or in combination with other plasmids, to provide transformedcorn plants, using transformation methods as described below toincorporate transgenes into the genetic material of the corn plant(s).

Expression Vectors for Corn Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linkedto a regulatory element (a promoter, for example) that allowstransformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene, isolated from transposonTn5, which, when placed under the control of plant regulatory signals,confers resistance to kanamycin (Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983)). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin (Vanden Elzen et al., Plant Mol. Biol., 5:299(1985)).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, and aminoglycoside-3′-adenyltransferase, the bleomycin resistance determinant (Hayford et al., PlantPhysiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987),Svab et al., Plant Mol. Biol. 14:197 (1990), and Hille et al., PlantMol. Biol. 7:171 (1986)). Other selectable marker genes conferresistance to herbicides such as glyphosate, glufosinate or bromoxynil(Comai et al., Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell2:603-618 (1990) and Stalker et al., Science 242:419-423 (1988)).

Selectable marker genes for plant transformation that are not ofbacterial origin include, for example, mouse dihydrofolate reductase,plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactatesynthase (Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shahet al., Science 233:478 (1986), and Charest et al., Plant Cell Rep.8:643 (1990)).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells rather than directgenetic selection of transformed cells for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used genes for screeningpresumptively transformed cells include beta-glucuronidase (GUS),beta-galactosidase, luciferase, and chloramphenicol acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al.,EMBO J. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci. U.S.A. 84:131(1987), and DeBlock et al., EMBO J. 3:1681 (1984). Another approach tothe identification of relatively rare transformation events has been useof a gene that encodes a dominant constitutive regulator of the Zea maysanthocyanin pigmentation pathway (Ludwig et al., Science 247:449(1990)).

In vivo methods for visualizing GUS activity that do not requiredestruction of plant tissue are also available. However, these in vivomethods for visualizing GUS activity have not proven useful for recoveryof transformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

A gene encoding Green Fluorescent Protein (GFP) has been utilized as amarker for gene expression in prokaryotic and eukaryotic cells (Chalfieet al., Science 263:802 (1994)). GFP and mutants of GFP may be used asscreenable markers.

Expression Vectors for Corn Transformation: Promoters

Genes included in expression vectors must be driven by nucleotidesequence comprising a regulatory element, for example, a promoter.Several types of promoters are now well known in the transformationarts, as are other regulatory elements that can be used alone or incombination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain organs,such as leaves, roots, seeds and tissues such as fibers, xylem vessels,tracheids, or sclerenchyma. Such promoters are referred to as“tissue-preferred”. Promoters which initiate transcription only incertain tissue are referred to as “tissue-specific”. A “cell type”specific promoter primarily drives expression in certain cell types inone or more organs, for example, vascular cells in roots or leaves. An“inducible” promoter is a promoter which is under environmental control.Examples of environmental conditions that may effect transcription byinducible promoters include anaerobic conditions or the presence oflight. Tissue-specific, tissue-preferred, cell type specific, andinducible promoters constitute the class of “non-constitutive”promoters. A “constitutive” promoter is a promoter which is active undermost environmental conditions.

A. Inducible Promoters—An inducible promoter is operably linked to agene for expression in corn. Optionally, the inducible promoter isoperably linked to a nucleotide sequence encoding a signal sequencewhich is operably linked to a gene for expression in corn. With aninducible promoter the rate of transcription increases in response to aninducing agent. Any inducible promoter can be used in the instantinvention. See Ward et al., Plant Mol. Biol. 22:361-366 (1993).Exemplary inducible promoters include, but are not limited to, that fromthe ACEI system which responds to copper (Mett et al., Proc. Natl. Acad.Sci. U.S.A. 90:4567-4571 (1993)); In2 gene from maize which responds tobenzenesulfonamide herbicide safeners (Gatz et al., Mol. Gen. Genetics243:32-38 (1994)) or Tet repressor from Tn10 (Gatz et al., Mol. Gen.Genetics 227:229-237 (1991)). A particularly preferred induciblepromoter is a promoter that responds to an inducing agent to whichplants do not normally respond. An exemplary inducible promoter is theinducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone (Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991)).

B. Constitutive Promoters—A constitutive promoter is operably linked toa gene for expression in corn or the constitutive promoter is operablylinked to a nucleotide sequence encoding a signal sequence which isoperably linked to a gene for expression in corn. Many differentconstitutive promoters can be utilized in the instant invention.Exemplary constitutive promoters include, but are not limited to, thepromoters from plant viruses such as the 35S promoter from CaMV (Odellet al., Nature 313:810-812 (1985)) and the promoters from such genes asrice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin(Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensenet al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor.Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730(1984)) and maize H3 histone (Lepetit et al., Mol. Gen. Genetics231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3): 291-300(1992)).

The ALS promoter, Xba1/Nco1 fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/Nco1fragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530.

C. Tissue-specific or Tissue-preferred Promoters—A tissue-specificpromoter is operably linked to a gene for expression in corn.Optionally, the tissue-specific promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in corn. Plants transformed with a gene ofinterest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zml3or a microspore-preferred promoter such as that from apg (Twell et al.,Sex. Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondrion or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Knox, C., et al.,Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol. 91:124-129(1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuoka et al.,Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell. Biol.108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon, etal., Cell 39:499-509 (1984), Stiefel, et al., Plant Cell 2:785-793(1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is corn. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant inbred line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis Protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btalpha-endotoxin gene. Moreover, DNA molecules encoding alpha-endotoxingenes can be purchased from American Type Culture Collection, Manassas,Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and31998.

C. A lectin. See, for example, the article by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487. The application teaches the use of avidin and avidinhomologues as larvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus alpha-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Pratt et al., Biochem. Biophys. Res. Comm. 163:1243(1989) (an allostatin is identified in Diploptera puntata). See alsoU.S. Pat. No. 5,266,317 to Tomalski et al., who disclose genes encodinginsect-specific, paralytic neurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper-accumulation of a monoterpene, asesquiterpene, a steroid, a hydroxamic acid, a phenylpropanoidderivative or another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance).

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-beta, lytic peptide analog torender transgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. Rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect.

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo-alpha-1, 4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-alpha-1, 4-D-galacturonase. See Lamb et al., BioTechnology 10:1436 (1992). The cloning and characterization of a genewhich encodes a bean endopolygalacturonase-inhibiting protein isdescribed by Toubart et al., Plant J. 2:367 (1992).

R. A developmental-arrestive protein produced in nature by a plant. Forexample, Logemann et al., BioTechnology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes that Confer Resistance to an Herbicide, for Example:

A. An herbicide that inhibits the growing point or meristem, such as animidazolinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance conferred by mutant5-enolpyruvylshikimate-3-phosphate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase (PAT) and Streptomyceshygroscopicus PAT bar genes), and pyridinoxy or phenoxy propionic acidsand cyclohexones (ACCase inhibitor-encoding genes). See, for example,U.S. Pat. No. 4,940,835 to Shah, et al., which discloses the nucleotidesequence of a form of EPSP which can confer glyphosate resistance. A DNAmolecule encoding a mutant aroA gene can be obtained under ATCCaccession number 39256, and the nucleotide sequence of the mutant geneis disclosed in U.S. Pat. No. 4,769,061 to Comai. European patentapplication No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a PAT gene is provided in Europeanapplication No. 0 242 246 to Leemans et al. DeGreef et al.,BioTechnology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for PAT activity. Exemplary ofgenes conferring resistance to phenoxy propionic acids and cyclohexones,such as sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3genes described by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+genes) or a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes that Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knutzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992)

B. Increased resistance to high light stress such as photo-oxidativedamages, for example by transforming a plant with a gene coding for aprotein of the Early Light Induced Protein family (ELIP) as described inWO 03074713 in the name of Biogemma.

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bact. 170:810 (1988)(nucleotide sequence of Streptococcus mutants fructosyltransferasegene), Steinmetz et al., Mol. Gen. Genet. 20:220 (1985) (nucleotidesequence of Bacillus subtilis levansucrase gene), Pen et al.,BioTechnology 10:292 (1992) (production of transgenic plants thatexpress Bacillus lichenifonnis α-amylase), Elliot et al., Plant Molec.Biol. 21:515 (1993) (nucleotide sequences of tomato invertase genes),Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley alpha-amylase gene), and Fisher et al., PlantPhysiol. 102:1045 (1993) (maize endosperm starch branching enzyme II).

D. Increased resistance/tolerance to water stress or drought, forexample, by transforming a plant to create a plant having a modifiedcontent in ABA-Water-Stress-Ripening-Induced proteins (ARS proteins) asdescribed in WO 0183753 in the name of Biogemma, or by transforming aplant with a nucleotide sequence coding for a phosphoenolpyruvatecarboxylase as shown in WO02081714. The tolerance of corn to drought canalso be increased by an overexpression of phosphoenolpyruvatecarboxylase (PEPC-C4), obtained, for example from sorghum.

E. Increased content of cysteine and glutathione, useful in theregulation of sulfur compounds and plant resistance against variousstresses such as drought, heat or cold, by transforming a plant with agene coding for an Adenosine 5′ Phosphosulfate as shown in WO 0149855.

F. Increased nutritional quality, for example, by introducing a zeingene which genetic sequence has been modified so that its proteinsequence has an increase in lysine and proline. The increasednutritional quality can also be attained by introducing into the maizeplant an albumin 2S gene from sunflower that has been modified by theaddition of the KDEL peptide sequence to keep and accumulate the albuminprotein in the endoplasmic reticulum.

G. Decreased phytate content: 1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene. 2) A gene could be introduced thatreduced phytate content. In maize, this, for example, could beaccomplished, by cloning and then reintroducing DNA associated with thesingle allele which is responsible for maize mutants characterized bylow levels of phytic acid. See Raboy et al., Maydica 35:383 (1990).

4. Genes that Control Male Sterility

A. Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN-Ac-PPT. See international publication WO 01/29237.

B. Introduction of various stamen-specific promoters. See internationalpublications WO 92/13956 and WO 92/13957.

C. Introduction of the barnase and the barstar genes. See Paul et al.,Plant Mol. Biol. 19:611-622, 1992).

The laborious, and occasionally unreliable, detasseling process can beavoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMSinbred are male sterile as a result of factors resulting from thecytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent inmaize plants, since only the female provides cytoplasm to the fertilizedseed. CMS plants are fertilized with pollen from another inbred that isnot male-sterile. Pollen from the second inbred may or may notcontribute genes that make the hybrid plants male-fertile. Seed fromdetasseled fertile maize and CMS produced seed of the same hybrid can beblended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar, et al. and chromosomal translocationsas described by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511.These and all patents referred to are incorporated by reference.

There are many other methods of conferring genetic male sterility in theart, each with its own benefits and drawbacks. These methods use avariety of approaches such as delivering into the plant a gene encodinga cytotoxic substance associated with a male tissue specific promoter oran antisense system in which a gene critical to fertility is identifiedand an antisense to that gene is inserted in the plant (see,Fabinjanski, et al., EPO 89/3010153.8, Publication No. 329,308 and PCTApplication PCT/CA90/00037, published as WO 90/08828).

Another system useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see, Carlson, Glenn R., U.S. Pat. No. 4,936,904). Applicationof the gametocide, timing of the application and genotype specificityoften limit the usefulness of the approach.

Waxy Starch

The waxy characteristic is an example of a recessive trait. In thisexample, the progeny resulting from the first backcross generation (BC1)must be grown and selfed. A test is then run on the selfed seed from theBC 1 plant to determine which BC 1 plants carried the recessive gene forthe waxy trait. In other recessive traits additional progeny testing,for example, growing additional generations such as the BC1S1, may berequired to determine which plants carry the recessive gene.

Tissue Cultures and In Vitro Regeneration of Corn Plants

A further aspect of the invention relates to tissue cultures of the cornplant designated I9545. As used herein, the term “tissue culture”indicates a composition comprising isolated cells of the same or adifferent type or a collection of such cells organized into parts of aplant. Exemplary types of tissue cultures are protoplasts, calli, andplant cells that are intact in plants or parts of plants, such asembryos, pollen, flowers, kernels, ears, cobs, leaves, husks, stalks,roots, root tips, anthers, silk, and the like. In a preferredembodiment, the tissue culture comprises embryos, protoplasts,meristematic cells, pollen, leaves, or anthers derived from immaturetissues of these plant parts. Means for preparing and maintaining planttissue cultures are well known in the art (U.S. Pat. Nos. 5,538,880 and5,550,318, each incorporated herein by reference in their entirety). Byway of example, a tissue culture comprising organs such as tassels oranthers has been used to produce regenerated plants (U.S. Pat. Nos.5,445,961 and 5,322,789, the disclosures of which are incorporatedherein by reference).

One type of tissue culture is tassel/anther culture. Tassels containanthers which in turn enclose microspores. Microspores develop intopollen. For anther/microspore culture, if tassels are the plantcomposition, they are preferably selected at a stage when themicrospores are uninucleate, that is, include only one, rather than twoor three nuclei. Methods to determine the correct stage are well knownto those skilled in the art and include mitramycin fluorescent staining(Pace, et al. (1987)), trypan blue (preferred) and acetocarminesquashing. The mid-uninucleate microspore stage has been found to be thedevelopmental stage most responsive to the subsequent methods disclosedto ultimately produce plants.

Examples of processes of tissue culturing and regeneration of corn aredescribed in, for example, European Patent Application 0 160 390; Greenand Rhodes (1982); Duncan, et al. (1985); Songstad, et al. (1988); Rao,et al. (1986); Conger, et al. (1987); PCT Application WO 95/06128;Armstrong and Green (1985); Gordon-Kamm, et al. (1990); and U.S. Pat.No. 5,736,369.

Duncan, Williams, Zehr, and Widholm, Planta 165:322-332 (1985) reflectsthat 97% of the plants cultured that produced callus were capable ofplant regeneration. Subsequent experiments with both inbreds and hybridsproduced 91% regenerable callus that produced plants. In a further studyin 1988, Songstad, Duncan and Widholm in Plant Cell Reports 7:262-265(1988), reports several media additions that enhance regenerability ofcallus of two inbred lines. Other published reports also indicated that“nontraditional” tissues are capable of producing somatic embryogenesisand plant regeneration. K. P. Rao, et al., Maize Genetics CooperationNewsletter, 60:64-65 (1986), refers to somatic embryogenesis from glumecallus cultures and B. V. Conger, et al., Plant Cell Reports, 6:345-347(1987) indicates somatic embryogenesis from the tissue cultures of maizeleaf segments. Thus, it is clear from the literature that the state ofthe art is such that these methods of obtaining plants are, and were,“conventional” in the sense that they are routinely used and have a veryhigh rate of success.

Tissue culture of maize is described in European Patent Application,Publication 160,390, incorporated herein by reference. Maize tissueculture procedures are also described in Green and Rhodes, “PlantRegeneration in Tissue Culture of Maize,” Maize for Biological Research,Plant Molecular Biology Association, Charlottesville, Va., pp. 367-372(1982) and in Duncan, et al., “The Production of Callus Capable of PlantRegeneration from Immature Embryos of Numerous Zea mays Genotypes,” 165Planta 322-332 (1985). Thus, another aspect of this invention is toprovide cells which upon growth and differentiation produce maize plantshaving the physiological and morphological characteristics of inbredcorn line I9545. In one embodiment, cells of inbred corn line I9545 aretransformed genetically, for example with one or more genes describedabove, for example by using a transformation method described in U.S.application Ser. No. 09/042,426, incorporated herein by reference, andtransgenic plants of inbred corn line I9545 are obtained and used forthe production of hybrid maize plants.

The present invention provides a genetic complement of the corn plantvariety designated I9545. As used herein, the phrase “geneticcomplement” means an aggregate of nucleotide sequences, the expressionof which defines the phenotype of a corn plant or a cell or tissue ofthat plant. By way of example, a corn plant is genotyped to determine arepresentative sample of the inherited markers it possesses. Markers arealleles at a single locus. They are preferably inherited in codominantfashion so that the presence of both alleles at a diploid locus isreadily detectable, and they are free of environmental variation, i.e.,their heritability is 1. This genotyping is preferably performed on atleast one generation of the descendant plant for which the numericalvalue of the quantitative trait or traits of interest are alsodetermined. The array of single locus genotypes is expressed as aprofile of marker alleles, two at each locus. The marker alleliccomposition of each locus can be either homozygous or heterozygous.Homozygosity is a condition where both alleles at a locus arecharacterized by the same nucleotide sequence or size of a repeatedsequence. Heterozygosity refers to different conditions of the gene at alocus. A preferred type of genetic marker for use with the invention issimple sequence repeats (SSRs), although potentially any other type ofgenetic marker could be used, for example, restriction fragment lengthpolymorphisms (RFLPs), amplified fragment length polymorphisms (AFLPs),single nucleotide polymorphisms (SNPs), and isozymes.

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of the foregoing illustrative embodiments, itwill be apparent to those of skill in the art that variations, changes,modifications, and alterations may be applied to the composition,methods, and in the steps or in the sequence of steps of the methodsdescribed herein, without departing from the true concept, spirit, andscope of the invention. More specifically, it will be apparent thatcertain agents that are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope, and concept of the invention as defined by theappended claims.

Industrial Uses

The present invention therefore also discloses an agricultural productcomprising a plant of the present invention or derived from a plant ofthe present invention. The present invention also discloses anindustrial product comprising a plant of the present invention orderived from a plant of the present invention. The present inventionfurther discloses methods of producing an agricultural or industrialproduct comprising planting seeds of the present invention, growingplants from said seeds, harvesting the plants and processing the plantsto obtain an agricultural or industrial product.

Maize is used as human food, livestock feed, and as raw material inindustry. Dent corn is usually used as livestock feed, but can also beused to make many food and industrial products. The food uses of maize,in addition to human consumption of maize kernels, also include bothproducts of dry- and wet-milling industries. The principal products ofmaize dry milling are grits, meal and flour. The maize wet-millingindustry can provide maize starch, maize syrups, and dextrose for fooduse. Maize oil is recovered from maize germ, which is a by-product ofboth dry- and wet-milling industries.

Maize, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs, and poultry. Industrial uses of maize include productionof ethanol, maize starch in the wet-milling industry and maize flour inthe dry-milling industry. The industrial applications of maize starchand flour are based on functional properties, such as viscosity, filmformation, adhesive properties, and ability to suspend particles. Themaize starch and flour have application in the paper and textileindustries. Other industrial uses include applications in adhesives,building materials, foundry binders, laundry starches, explosives,oil-well muds, and other mining applications. Plant parts other than thegrain of maize are also used in industry: for example, stalks and husksare made into paper and wallboard and cobs are used for fuel and to makecharcoal.

The seed of inbred corn line I9545, the plant produced from the inbredseed, the hybrid corn plant produced from the crossing of the inbred,hybrid seed, and various parts of the hybrid corn plant and transgenicversions of the foregoing, can be utilized for human food, livestockfeed, and as a raw material in industry.

Table(s)

Inbred corn line I9545 can be used as a breeding line for creating newhybrid corn varieties. Table 2 shows comparisons of data collected fromhybrids created using inbred tester lines crossed to either inbred cornline I9545 as a parent or another corn line as a parent. The inbredlisted in the Tester column is the tester line crossed to either I9545or another line for comparison purposes. For example, in the first dataset, I9545 and MBS8814 were each crossed with the same four testers. Theresults allow one to compare I9545 and MBS8814. In the other two datasets, I9545 can be compared with TR6331 and PCW. The entire data set canalso be viewed as describing I9545 hybrids with other, relatedcommercial hybrids. In Table 2, column 1 shows the tester line, column 2shows the inbred line, column 3 shows the number of locations (# Loc),column 4 shows the yield in bushels/acre (Yld), column 5 shows thepercent water (% H2O), column 6 shows the yield to moisture ratio (Y/M),column 7 shows the percent stalk lodging (% SL), column 8 shows thepercent root lodging (% RL), column 9 shows the test weight inpounds/bushel (TW), column 10 shows the plant height in inches (PHT),and column 11 shows the ear height in inches (EHT).

TABLE 2 # % % % Tester Inbred Loc Yld H₂O Y/M SL RL TW PHT EHT TR7245HXTI9545 28 201.7 21.8 9.3 1.3 2.4 55.1 117 54 GP280 I9545 12 219.5 24.09.2 0.7 12.5 54.7 121 55 MBS2623 I9545 11 208.5 21.5 9.7 4.1 3.4 55.0102 40 TR8453 I9545 13 218.1 24.1 9.0 0.5 3.6 54.9 112 51 I9545 Average212.0 22.9 9.3 1.7 5.5 54.9 113 50 TR7245HXT MBS8814 28 229.2 22.7 10.11.6 1.2 54.5 124 58 GP280 MBS8814 12 221.8 24.2 9.2 4.0 16.8 54.7 131 60MBS2623 MBS8814 11 209.2 21.7 9.6 3.7 2.2 55.0 106 42 TR8453 MBS8814 13231.6 25.0 9.3 2.7 0.9 54.4 119 61 MBS8814 Average 223.0 23.4 9.6 3.05.3 54.7 120 55.25 TR7245HXT I9545 28 201.7 21.8 9.3 1.3 2.4 55.1 117 54TR6467GTCBLLRW I9545 6 213.0 23.7 9.0 2.2 0.0 54.5 TR8453 I9545 13 218.124.1 9.0 0.5 3.6 54.9 112 51 I9545 Average 210.9 23.2 9.1 1.3 2.0 54.8114.5 52.5 TR7245HXT TR6331 28 205.7 19.7 10.4 3.1 0.7 55.9 121 56TR6467GTCBLLRW TR6331 6 197.5 19.2 10.3 0.8 0.0 56.2 TR8453 TR6331 13211.6 23.4 9.0 2.7 0.4 54.9 109 53 TR6331 Average 204.9 20.8 9.9 2.2 0.455.7 115 54.5 Line “A” I9545 9 210.5 23.4 9.0 0.8 2.6 54.6 110 42 Line“A” PCW 9 206.0 21.3 9.7 2.3 3.2 55.1 110 43

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the invention and does not pose a limitation on the scope ofthe invention unless otherwise claimed. No language in the specificationshould be construed as indicating any non-claimed element as essentialto the practice of the invention.

Deposit Information

A deposit of the BECK′S SUPERIOR HYBRIDS proprietary INBRED CORN LINE19545 disclosed above and recited in the appended claims has been madewith the American Type Culture Collection (ATCC), 10801 UniversityBoulevard, Manassas, Va. 20110. The date of deposit was March 20, 2013.The deposit of 2,500 seeds was taken from the same deposit maintained byBECK′S SUPERIOR HYBRIDS since prior to the filing date of thisapplication. All restrictions will be irrevocably removed upon grantingof a patent, and the deposit is intended to meet all of the requirementsof 37 C.F.R. §§1.801-1.809. The ATCC Accession Number is PTA-13633. Thedeposit will be maintained in the depository for a period of thirtyyears, or five years after the last request, or for the enforceable lifeof the patent, whichever is longer, and will be replaced as necessaryduring that period.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions, and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

What is claimed is:
 1. A seed of inbred corn line I9545, wherein arepresentative sample of seed of said inbred corn line was depositedunder ATCC Accession No. PTA-13633.
 2. A plant, or a part thereof,produced by growing the seed of claim
 1. 3. A tissue culture of cells ofthe plant of claim
 2. 4. The tissue culture of claim 3, wherein cells ofthe tissue culture are from a tissue selected from the group consistingof leaf, pollen, embryo, root, root tip, anther, silk, flower, kernel,ear, cob, husk, stalk and meristem.
 5. A corn plant regenerated from thetissue culture of claim 4, wherein the regenerated plant has all of themorphological and physiological characteristics of inbred corn lineI9545 as listed in Table
 1. 6. The seed of claim 1, wherein said seedfurther comprises a transgene.
 7. The seed of claim 6, wherein thetransgene confers a trait selected from the group consisting of malesterility, herbicide tolerance, insect resistance, disease resistance,waxy starch, modified fatty acid metabolism, modified phytic acidmetabolism, modified carbohydrate metabolism and modified proteinmetabolism.
 8. The seed of claim 6, wherein said seed comprises a singlelocus conversion.
 9. The seed of claim 8, wherein the single locusconversion confers a trait selected from the group consisting of malesterility, herbicide tolerance, insect resistance, disease resistance,waxy starch, modified fatty acid metabolism, modified phytic acidmetabolism, modified carbohydrate metabolism and modified proteinmetabolism.
 10. A method of producing hybrid corn seed comprisingcrossing the plant of claim 2 with a different inbred corn line andharvesting the resultant hybrid corn seed.
 11. A hybrid corn seedproduced by the method of claim
 10. 12. A hybrid corn plant, or a partthereof, produced by growing said hybrid seed of claim
 11. 13. A methodof introducing one or more desired traits into inbred corn line I9545,wherein the method comprises: (a) crossing an inbred corn line I9545plant, wherein a representative sample of seed of said plant wasdeposited under ATCC Accession No. PTA-13633, with a plant of anothercorn line that comprises a desired trait to produce progeny plants; (b)selecting one or more progeny plants that have the desired trait; (c)backcrossing selected progeny plants with inbred corn line I9545 plantsto produce backcross progeny plants; (d) selecting for backcross progenyplants that have the desired trait(s); and (e) repeating steps (c) and(d) one or more times in succession to produce selected second or higherbackcross progeny plants that comprise the desired trait.
 14. The methodof claim 13, wherein the desired trait is selected from the groupconsisting of male sterility, herbicide tolerance, insect resistance,disease resistance, waxy starch, modified fatty acid metabolism,modified phytic acid metabolism, modified carbohydrate metabolism andmodified protein metabolism.
 15. A corn plant produced by the method ofclaim 14, wherein the plant has the desired trait(s) and all of themorphological and physiological characteristics of inbred corn lineI9545 as listed in Table
 1. 16. The plant of claim 15, wherein thedesired trait is herbicide resistance and the resistance is conferred toan herbicide selected from the group consisting of imidazolinone,sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine andbenzonitrile.
 17. The plant of claim 15, wherein the desired trait isinsect resistance and the insect resistance is conferred by a transgeneencoding a Bacillus thuringiensis endotoxin.
 18. The plant of claim 15,wherein the desired trait is modified fatty acid metabolism or modifiedcarbohydrate metabolism and said desired trait is conferred by a nucleicacid encoding a protein selected from the group consisting offructosyltransferase, levansucrase, alpha-amylase, invertase and starchbranching enzyme or DNA encoding an antisense of stearyl-ACP desaturase.19. A method of producing a hybrid corn seed, wherein the methodcomprises crossing inbred corn line I9545, wherein said inbred corn linehas been genetically modified to add a desired trait, with a differentcorn plant and-harvesting the resultant hybrid corn seed.
 20. A hybridcorn seed produced by the method of claim
 19. 21. A hybrid corn plant,or a part thereof, produced by growing said hybrid seed of claim
 20. 22.A method for obtaining an inbred corn line comprising: (a) planting acollection of seed comprising seed of a corn hybrid, one of whoseparents is a plant according to claim 2, or a corn plant having all thephysiological and morphological characteristics of a plant according toclaim 2, said collection of seed also comprising seed of said inbredcorn line I9545; (b) growing plants from said collection of seed; (c)identifying said inbred plants; (d) selecting said inbred plants; and(e) controlling pollination in a manner which preserves the homozygosityof said inbred plants.
 23. The method according to claim 22, whereinsaid one parent has essentially all the physiological and morphologicalcharacteristics of inbred corn line I9545, seed of said line having beendeposited under ATCC Accession No. PTA-13633, and further comprises oneor more single gene transferred traits.
 24. A method comprisingintrogressing one or more single gene traits into inbred corn lineI9545, seed of said line having been deposited under ATCC Accession No.PTA-13633, using one or more markers for marker assisted selection amongcorn lines to be used in a corn breeding program, the markers beingassociated with said one or more single gene traits, wherein theresulting corn line has essentially all the physiological andmorphological characteristics of a plant of inbred corn line I9545 aslisted in Table 1 and further comprises said one or more single genetransferred traits.
 25. A method of producing a commodity plant product,comprising obtaining the plant of claim 2, or a part thereof, andproducing the commodity plant product from said plant or plant partthereof, wherein said commodity plant product is selected from the groupconsisting of livestock feed, starch, ethanol, biomass, biofuel andrefined chemicals.